The stapled peptide technology has afforded a novel method for the stabilization of biologically relevant peptide helices. Thus far, it has created unique opportunities for targeting discrete components of complex signaling pathways relevant to the pathogenesis of cancer. The use of this methodology has enabled our study of the apoptotic signaling pathway and, more recently, the manipulation of transcriptional pathways restricted to the nucleus. We aim to significantly evolve the stapled peptide strategy through chemical refinement in order to expand our ability to target pathologic protein interactions implicated in cancer. Throughout the course of our research into the function of p53 family members in cancer (see ZIA BC 011376 project summary), we have found that while our HDM2/HDMX targeting compound SAH-p53-8 restores the transcriptional activity of inhibited p53, several other lines of activity have been found, some of which are entirely independent of p53. In order to provide a chemical approach to target identification, we have developed photactivatable alpha-helices of p53 (pSAH-p53s) which are capable of cross-linking covalently to their target proteins by excitation with UV light. To do this, we have designed our photoactivatable compounds using as a template the sequence of SAH-p53-8, our most active compound to date. Photochemical cross-linking is accomplished by incorporating into the sequence of the peptide a para-benzophenone phenylalanine residue in lieu of an amino acid putatively involved in the requisite protein-protein interaction. As a proof of principle study, we combined a pSAH-p53 with a mixture of its target protein HDM2 and a spectator protein in a superstoichiometric ratio. We found that after exposure to UV light, pSAH-p53 cross-links selectively to HDM2 despite the overwhelming presence of other proteins. With these data on hand, we then proceeded to evaluate the compounds in the complex setting of a cell extract. Gratifyingly, we have found that pSAH-p53s effectively cross-link HDM2 and HDMX from lysates. More importantly, we also found that the pSAH-p53s target other proteins as well. Current efforts are aimed at identifying these novel binding partners and determining their significance to p53 family pathways. Given our success in creating pSAH-p53s that selectively bind their targets and crosslink with them on exposure to UV light, we have carried out the synthesis of pSAH-p63 and pSAH-p73 peptides based on their respective TADs by solid phase peptide synthesis. As members of the same family, p63 and p73 share many structural similarities with p53, and much like p53, they have the ability to form multimeric complexes and bind to p53-responsive promoters. Despite this redundancy, there are functions of p63 and p73 in cells that are unique, playing roles in cellular motility, invasiveness and differentiation. Further, these two proteins also possess TADs of their own with cursory sequence homology. We have validated this strategy in vitro using both recombinant proteins and we are now investigating its utility in experiments carried out both using cell extracts and live cells. These experiments will provide a better understanding of the protein-protein interactions that drive p53, p63 and p73 function.